Re-introduction of an extinct population of Pulsatilla patens using different propagation techniques

The study focuses on the propagation of a rare and endangered plant species (Pulsatilla patens) to re-introduce an extinct population from calamine area in Southern Poland. The plants were propagated from seeds, rhizome cuttings, or regenerated in vitro from shoot tips, hypocotyls with roots or cotyledons of seedlings on Murashige & Skoog (MS) medium supplemented with 0.25 or 0.50 mg L−1 BAP (Benzylaminopurine) via direct and indirect organogenesis or somatic embryogenesis (SE). The most efficient micropropagation method was with shoot tips as an explant on MS + 0.25 mg L−1 BAP where 97% of the explants produced multiple shoots, mass SE was observed after transfer on ½ MS with 2% saccharose; 267 (35%) shoots rooted on ½ MS + 2% saccharose were acclimatized to ex vitro conditions. Flow cytometry revealed genome size stability of propagated plantlets. Low genetic differentiation between micropropagated plantlets and initial material was indicated by ISSR (Inter Simple Sequence Repeat) markers. Totally, 132 vigorous plantlets obtained on various pathways were introduced to the field plots in 2020; 30.33% survived the winter, and several reached the generative stage and flowered in the spring 2021. In next season (March/April 2022) the number of introduced plants decreased to 25% while the number of flowering and fruiting shoots in different clumps increased in some plots. This is the first report of successful re-introduction of the endangered P. patens based on micropropagation, rhizome cuttings, and seed germination.

and half-open forests. The species was formerly abundant at many metalliferous (Zn-Pb rich; calamine) sites in southern Poland, e.g. in Bolesław, Stary Olkusz and Jaworzno 20,21 ; currently not a single plant remained in this area (monitoring in 2019). The active protection and restoration of populations in calamine grasslands was highlighted recently because metallicolous vegetation forms unique assemblages of a limited number of metallophyte species that successfully colonized these sites. These assemblages are the result of microevolutionary processes in stressful environments, and there is only a limited number of sites where they can be observed 22,23 . Metallophyte species are also unique resources that could be exploited for the development of phytoremediation including revegetation 24 . Calamine grasslands benefit from conservation programs and are considered as a threaten habitat type that is legally protected by European (European Habitats Directive 92/43/EEC, Appendix I, habitat type 6130), national or regional laws. Among sites of the Natura 2000 network, 118 are designated for this habitat type 25 . Techniques of multiplication and cryopreservation-the hope for maintaining the genetic pool of pasque-flower. The preservation of endangered, threatened plant species by ex situ conservation, including in vitro culture and cryopreservation, prevents losses of plant biodiversity 26 , recovers genetic diversity from preserved material, and allows transplantation of multiplied plants to botanical gardens and field sites [27][28][29][30] .
In vitro culture serves as an excellent biotechnological tool for ex situ conservation of different endangered species 31,32 , including Pulsatilla vulgaris 33 , and also species growing at metalliferous sites [34][35][36] . However, the reintrodution of in vitro propagated plants to the field sites, will be successful provided the areas are properly managed before the transplantation 22,25,37 .
The main goals of the present study was to restore an extinct calamine grassland population of the endangered P. patens by using plants propagated from seeds, rhizome cuttings, or micropropagated in vitro, and to monitor their successful acclimatization to the field conditions. Molecular markers and flow cytometry were used to assess the genetic quality of the in vitro regenerated plantlets.

Results
Cytological characteristic of plants. Pulsatilla  Poor seed germination and plantlets development from rhizome cuttings. For seed germination tests, 1022 fruits were used (Table 1). Germination and seedlings development depended on the substrate. The highest germination (73%) was on ½ MS (Murashige & Skoog) with 2% saccharose, very low in garden soil (17%), and deacidified peat mixed with sand (18%). Five plants recovered in spring 2020 from rhizome cuttings were grown in a common garden until transplantation to Sadowa Góra in October 2020.
Organogenesis and somatic embryogenesis, rooting and acclimatization as a method of restoring population. Direct and indirect (via callus) organogenesis (adventitious shoot development) was induced in all used explants, shoot tip, cotyledon, hypocotyl + root (Fig. 1D,E) on MS medium supplemented with BAP (Benzylaminopurine) in concentrations 0.25 or 0.50 mg L −1 . Two-way ANOVA showed significant differences between types of explants but not between the media supplemented with different concentrations of BAP (MS + 0.25 or 0.50 mg L −1 BAP). Interaction between type of explants and medium was also non-significant. Tukey's HSD test post-hoc confirmed significant differences between hypocotyl + root on MS + 0.25 mg L −1 BAP and shoot tips ( Table 2). Shoot tips excised from seedlings were the best explants. Altogether 91% responded for culture conditions, which was a much higher frequency compared to hypocotyl + root (64%) and cotyledon (7%). The most abundant adventitious shoot induction on one explant (several dozen, mass of shoots, uncountable) was observed on shoot tip explants on both growth media. Direct organogenesis was confirmed on histological sections, shoots developed from meristematic centers induced inside the explants (Fig. 1G-I). Rooting of adventitious shoots after transfer on ½ MS hormone-free medium (supplemented with 2% saccharose) was poor; out of 758 shoots obtained in three experiments, only 267 (35%) developed roots (Table 3, Fig. 1F). Rooting time was long and lasted up to several months without passaging the shoots on fresh medium.
Somatic embryos (SE) were induced on shoot tips and cotyledons ( Table 2, Fig. 1J) after explants transfer from inducing medium (MS + 0.25 or 0.50 mg L −1 BAP) onto ½ MS (2% saccharose); the 52 largest plants developed from SE (Fig. 1K), and 267 rooted shoots (Table 3) were moved to ex vitro conditions, transferred to pots filled with deacidified peat mixed with sand ( Fig. 1L-N) and they grew first indoor and then under garden conditions till transfer to the field site.
Genetic compatibility of regenerated plants with initial material. NeighborNet analysis showed some diversity among plants from natural population in Łagiewniki near Busko-Zdrój (Ł), which clustered in two groups together with plants derived from the seeds from this population (BZ). The most divergent group (bootstrap 81.7) contained plants Ł2 and Ł7 along with BZ3 seedling derived from Ł2. Most of the other plants were grouped in large second cluster together with regenerated plants (R1-R9), which exhibited low divergence and non-significant grouping (branch support < 50) (Fig. 2). Seedlings likely originated from seeds of different initial plants: BZ3 from Ł2; BZ6 from Ł6; BZ9 and BZ7 from Ł9. Regenerated plantlets (R1-R9) were genetically close to initial plants BZ1, BZ2, BZ4 (Fig. 2). NeighborNet analysis showed non-significant genetic diversity   Fig. 1). Genome size for analyzed diploid Pulsatilla species (2n = 2x = 16) varied from 8.80 to 11.42 pg/2C (Table 4). P. slavica was a tetraploid with 2n = 4x = 32 and possessed the largest genome (25.13 pg/2C). For P. patens, mean genome size was 2C = 10.93 pg. Detected differences between regenerated plants and the initial material as well as plants from the natural population Łagiewniki near Busko-Zdrój, and two other locations were statistically    www.nature.com/scientificreports/ field site in 15 October 2020 ( Fig. 3A-D). Before transplantation, six plots were selected in this area with varying degrees of shade (Table 5), on which propagated plants were dug into. Out of 44 plants micropropagated in vitro, 30 were introduced to the field site, and the remaining 14, that were too small for introduction to the field, were stored under greenhouse conditions until the following season. In April 2021, the monitoring of all six plots on Sadowa Góra revealed that out of 132 transplanted P. patens, 40 (30.33%) had survived the first winter, and some developed flowers (Fig. 3D right insert) and set seeds (Table 5). In next season (March/April 2022) the number of surviving individuals decreased to 25.00% while the number of flowering and fruiting shoots in different clumps increased in some plots, even to over 20 (plots 2, 6; Table 5). Fruits developed well and were filled with seeds (Table 5).

Discussion
The research objectives of this study were successfully achieved, including a completed life cycle of P. patens starting with sowing seeds into various substrates for plant multiplication, in vitro regeneration of plantlets, acclimatization of multiplied plants to the field conditions, re-introduction to the calamine grassland, and finally monitoring the frequency of plants that survived and developed flowers, fruits and set seeds in the following seasons. If the appropriate habitat is maintained in the following years, the recovered extinct population will contribute to the preservation of biodiversity of calamine grasslands 42 .  www.nature.com/scientificreports/ insects, mainly bees 45 . As the separation of the male and female sexual phases in the flower is not complete, selfpollination might occur, however, very rarely 44 . The production of fruits/seeds is high, since each flower might produce on average > 100 monospermed achenes 17 . Seeds retain viability and germination capacity for several years, thus forming a transient seed bank 7 . Hence the factors limiting germination and seedling development are the exact timing in late autumn the first year or early spring of the following year; critical factors are humidity and low temperatures and the thickness and compactness of the moss cover and the amount of accumulated litter 46 . For the persistence of the population it is necessary to maintain a relatively large diversity of microhabitats, that reduce seedling mortality, e.g. due to desiccation 9,17 . These factors were considered when preparing the area on Sadowa Góra for the re-introduction of P. patens multiplied plants. Six plots were selected with different degrees of canopy openness and slope within a historical forest, where some trees had been earlier removed ( Table 5). The survival rate of plants introduced to six plots on Sadowa Góra seems to be dependent on several factors. The most important is the developmental stage of introduced plants-juvenile with 2-3 leaves vs larger with several well-developed leaves. The survived individuals were introduced as one-or two-year-old plants originated from seeds or rhizome cuttings which allow us to conclude that the multiplied plant material must be grown at least two seasons in the field/experimental garden conditions before the introduction. The positive correlation between the insolation of the site and clumps growth/generative organ development was noticed. At completely exposed plots no. 2 and 6, not shaded by Scots pine, the clumps expanded and developed even dozens flowering/ fruiting branches (Table 5). In open space pollinator access to flowers must have been easier as evidenced by the development of fruits at these sites.
Good practices of introducing pasque-flower fulfilled. Several Pulsatilla species, e.g. P. grandis, P. koreana 47 , P. patens 12,47 , P. pratensis [48][49][50] , P. turczaninovii 51 , P. vernalis and P. vulgaris 33 , were multiplied by in vitro organogenesis and/or somatic embryogenesis, acclimatized to ex vitro and to semi-natural conditions. However, in none of these experiments, regenerated plants were re-introduced to the natural (extinct) populations, and their genetic uniformity was not confirmed with the initial material. It is well-known that in vitro conditions generate genome alteration (gene/genome mutations, epigenetic changes) of regenerated plants leading to the formation of new genotypes (e.g. aneuploids, polyploids). The introduction of such somaclonal variants into natural populations could disrupt the genetic structure of the population and even lead to the elimination of existing genotypes, as discussed by Żabicki et al. 31 . The genetic diversity of in vitro regenerated P. patens plants estimated in our research by ISSR molecular markers fell within the range of natural populations of this species. Plants multiplied in vitro and introduced into the field did not differ genetically from the initial seedlings and can be treated as one line (genotype). All regenerants represented the same ploidy level/genome size as the initial material and individuals from natural populations.
Our study concerned the re-introduction of the extinct population on Sadowa Góra (S Poland), where in the past P. patens was common in a large calamine grassland. Plants used for introduction were multiplied from the initial material originated from another (not adjacent) region of S Poland. We had herbarium specimens from the calamine grasslands, unfortunately, the quality of the isolated DNA was insufficient for ISSR analysis to show genetic uniformity of the multiplied material with the calamine extinct population. Based on low genetic differentiation of P. patens found in different areas of its range indicating higher levels of variation within populations than between populations 13 , we assumed that the genotypes obtained via multiplication of the material from S Poland population Łagiewniki near Busko-Zdrój is not genetically different from the extinct population on Sadowa Góra. To confirm this, we studied individuals from north-eastern Poland (Kolimagi near Kolno) using ISSR analysis. The results showed that there was no significant genetic difference between plants from Łagiewniki near Busko-Zdrój and Kolimagi near Kolno plants, and the rest of the samples, including regenerants. Also, twoyear-old plants obtained from seeds collected at Kolimagi near Kolno and transplanted to Sadowa Góra were well adapted to their climatic and habitat conditions. This suggests that we did not introduce aberrant genotypes of P. patens into the place of the extinct population. The transplanted plants reproduced sexually producing fully viable pollen and set seeds. Future perspectives. Micropropagation protocol of P. patens developed in our studies makes it possible to preserve the genetic pool of an endangered species by cryopreservation. The next step in conservation practice is developing an effective procedure for cryopreservation of shoot tips, shoots or somatic embryos of P. patens and introducing them for long-term storage in liquid nitrogen, including individuals from different European populations. Although several rare, endemic and endangered species have already been introduced to cryogenic conditions 32,52-54 , thousands still need to be cryopreserved, including all Pulsatilla species. It should be emphasized that in situ protection alone of endangered species will not guarantee their conservation, and thus must be supported by ex situ management with the use of biotechnological techniques and optionally seed banking as complementary for plant conservation including short-, medium-and long-term strategies 54 .

Conclusions
The breakthrough of this study is successful multiplication of the endangered species P. patens using different techniques, including in vitro regeneration via organogenesis and somatic embryogenesis, acclimatization to the field conditions of multiplied plants and their transplantation into the nature. Our studies could serve as a model for restoring extinct populations and preserving the biodiversity of rare species. Seed germination test, seedlings development, vegetative propagation. Mature fruits with seeds of P. patens, collected randomly from at least 15 plants in Łagiewniki near Busko-Zdrój, were germinated on different substrates, i.e. under sterile in vitro conditions on Petri dishes filled in with ½ MS 38 medium with 2% saccharose solidified with 0.8% agar (w/v) (pH 5.7-5.8), in garden soil (pH 6.0-6.5) or in deacidified peat (pH 5.5-6.5) mixed with sand (3:1 v/v) in pots. Fruits harvested (at the same manner as in Łagiewniki near Kolno Natura 2000 site "Sasanki w Kolimagach" PLH200025, NE Poland) were germinated in garden soil (pH 6.0-6.5). Fruit pappus-hairs were removed from each fruit with scissors or tweezers. For in vitro germination, fruits were sterilized according to Żabicka et al. 33 without any other pretreatment. Germination procedure was performed in triplicate.
Seedlings obtained from seeds germinated in vitro on ½ MS supplemented with 2% saccharose and transplanted to pots filled with peat mixed with sand (3:1 v/v) or germinated directly in garden soil in pots were acclimatized. Depending on the time of sowing the seeds, sprouting was directly in outdoor garden conditions (in spring), or first the seedlings were acclimatized indoors several weeks/months (late summer-winter) and then, in spring, transferred to the garden. Acclimatized seedlings were introduced on Sadowa Góra in Jaworzno in October 2020. At the time of planting on Sadowa Góra, the plants were one-or two-year-old.
Two mature patches of P. patens that had been dug up in the natural population Kolno Natura 2000 "Sasanki w Kolimagach" in June 2018 were transferred to garden conditions. In late November, when plants were dormant, their rhizomes were divided into several parts, moved to pots with garden soil and left till following season.  59 . Nuclear DNA content in at least 5000 nuclei per sample was estimated using a CyFlow SL Green (Partec GmbH, Münster, Germany) flow cytometer. Histograms were evaluated using a FlowMax program (Partec GmbH). The coefficient of variation (CV) of the G 0 /G 1 peak of Pulsatilla ranged from 2.51 to 4.68%. Nuclear DNA content was calculated using the linear relationship between the ratio of the 2C peak positions of Pulsatilla/Secale on a histogram of fluorescence intensities. The number of biological replications varied from 2 to 16. Low number of analyzed plants of some entries from natural sites was due to the restrictions of the regional Directorate for Environmental Protection or a small number of individuals in the population.  (Table 1) were used for molecular analysis. CTAB extraction method 60 was used for DNA isolation. Quality of DNA was checked on 1% agarose gel. The analysis of genetic diversity using five primers of ISSR markers (Suppl. Table 1) 61,62 , and electrophoresis were based on protocols developed by Żabicka et al. 33 . Selected samples were used for test of repeatability of results. Results of analysis using ISSR markers of 27 samples, were captured with a MultiDoc-It™ Imaging System with VisionWorks LS Analysis Software (UVP, Upland, CA, USA, https:// www. labor techn ik. com/ en/ vision-works-ls/ analy sis-softw are). SplitsTree v. 4.6 63 was used for splitting phylogenetic network (NeighborNet) construction, based on Dice coefficient. Bootstrap was calculated on 1000 replicates. Acclimatization to field conditions and transplantation to the place of extinct population in Southern Poland. Pulsatilla patens plants propagated from seeds, rhizome cuttings or micropropagated in vitro were gradually acclimatized to outdoor conditions (transferred from indoor to garden conditions) in the 2019-2020 seasons. In October 2020, all plantlets with several well-developed leaves were transplanted to nature and landscape complex Sadowa Góra (Jaworzno, Southern Poland), where in the past (till the 1980s) P. patens formed a large population in calamine grassland. Here, six site types (plots) were used: (1) slightly recessed area located at the top of a small mound formed after calamine mining-area completely exposed; (2) adjacent to the excavation with SE exposure and a slope of approx. 10°; area completely exposed; (3) nearby the small excavation with NW exposure and an inclination of approx. 25°-area partially shaded; (4) next to the shallow excavation with exposure E and SE, and an inclination of approx. 10°-area partially shaded by Scots pine trees growing in the vicinity; (5) next to the shallow excavation with Scots pine in the centre-SE exposure and a slope of approx. 15°, and area partially shaded by Scots pine; and (6) next to the shallow excavation with SE exposure and an inclination of approx. 25°-area completely exposed.
Plants were introduced to the field under the permission of the mayor of the city Jaworzno within the project "Good practices for enhancing biodiversity and active protection of calamine grasslands in the Silesia-Cracow region BioGalmany". The site monitoring and the observation of introduced plants (number of survived individuals/clumps, flowering and fruiting of shoots in different clumps) were conducted in two subsequent seasons (March/April 2021/2022).